The objective of the proposal is to determine the potential mutagenic events involved in the replication of chemical carcinogen damaged DNA templates in cultured mammalian cells treated with reactive metabolites of the environmental carcinogen, benzo[a]pyrene. These studies will be carried out with excision-repair deficient Chinese hamster ovary cells (CHO) and with Simian virus 40 (SV40) growing in the permissive host cells, CV-1P (African green monkey kidney cells). The specific objectives of this proposal are: 1) to determine in (plus/minus) 7Beta, 8Alpha dihydroxy-9Alpha, 10Alpha-epoxy 7, 8, 9, 10 tetrahydrobenzo[a]pyrene (BPDEI) damaged CHO cells the mode of DNA replication at the replication fork on both leading and lagging strands of nascent DNA, 2) with the same objective study replication of BPDEI damaged SV40 virus in CV-1P cells, 3) to test the ability of a variety of metabolites of benzo[a]pyrene to induce a mutagenic mode of error-prone DNA repair in CV-1P cells which acts on UV or BPDEI damaged temperature sensitive mutant SV40 virus, and 4) to analyze the various SV40 revertants obtained from the mutagenicity studies for detectable DNA deletions or additions. DNA replication at the replication fork will be studied in BPDEI damaged synchronized CHO cells using pH step alkaline elution assay, agarose gel electrophoresis and benzoylated naphtholated diethylaminoethyl cellulose column chromatography. SV40 reactivation frequencies and mutation rates of UV irradiated or BPDEI damaged SV40 virus in CV-1P cells treated with various metabolites of benzo[a]pyrene will be studied with an early (tsA-58) temperature sensitive mutant of SV40. Analysis of SV40 revertants for detectable deletions or additions will be done by restriction enzyme mapping. Replication of BPDEI damaged SV40 in CV-1P cells will be studied utilizing neutral sucrose gradient and agarose slab gel electrophoresis of SV40 viral DNA replication intermediates. Experimental data suggest that covalent binding of a chemical mutagen or carcinogen to cellular DNA and replication of the damaged template are crucial events during mutagenesis and carcinogenesis. Therefore, it is of interest to study the process of DNA replication in cells damaged by the environmental carcinogen benzo[a]pyrene in order to understand the mechanism by which DNA information is altered through DNA replication.